Electrospray ionization multi-stage mass spectrometry (ESI-MS(n)) was performed to study the fragmentation behaviour of seventeen steroidal alkaloids (4 protoverine-type alkaloids, 10 germine-type alkaloids and 3 zygadenin-type alkaloids) from the Chinese herb Veratrum nigrum L. The MS(n) spectra of the [M+H](+) ions for steroidal alkaloids provided a wealth of structural information on the substituted groups. In positive ion mode, the three types of alkaloids showed very different characteristic ions: m/z 436 or 418 for protoverine-type alkaloids; m/z 438, 420 or 402 for germine-type alkaloids; m/z 440 or 422 for zygadenin-type alkaloids. These fragments were used to deduce their mass spectral fragmentation mechanisms. Furthermore, the primary compounds in methanolic extracts of the herb of Veratrum nigrum L. were investigated by using liquid chromatography (LC)/ESI-MS(n). As a result, 21 steroidal alkaloids (5 protoverine-type alkaloids, 14 germine-type alkaloids and 2 zygadenin-type alkaloids) were selectively identified from 27 determined peaks. Eleven compounds were unambiguously identified by comparing with standard compounds and ten compounds were tentatively identified or deduced according to their MS(n) data. Two of these compounds (xingangermine and deacetyl xinganveratrine) were found to be novel steroidal alkaloids. In addition, the chemical structures of two pairs of steroidal alkaloid isomers were deduced by comparing their fragment ions. Given the important structural information of known and unknown steroidal alkaloids in crude herbal extracts, this study is useful for identifying these types of steroidal alkaloids in crude materials rapidly and selectively.